Chloroplasts are critical organelles that perceive and convey metabolic and stress signals to different cellular components, while remaining the seat of photosynthesis and a metabolic factory. The proteomes of intact leaves, chloroplasts, and suborganellar fractions of plastids have been evaluated in the model plant Arabidopsis, however fewer studies have characterized the proteomes of plastids in crops. Tomato (Solanum lycopersicum) is an important world-wide crop and a model system for the study of wounding, herbivory and fruit ripening. While significant advances have been made in understanding proteome and metabolome changes in fruit ripening, far less is known about the tomato chloroplast proteome or its subcompartments. With the long-term goal of understanding chloroplast proteome dynamics in response to stress, we describe a high-yielding method to isolate intact tomato chloroplasts and stromal proteins for proteomic studies. The parameters that limit tomato chloroplast yields were identified and revised to increase yields. Compared to published data, our optimized method increased chloroplast yields by 6.7- and 4.3-fold relative to published spinach and Arabidopsis leaf protocols, respectively; furthermore, tomato stromal protein yields were up to 79-fold higher than Arabidopsis stromal proteins yields. We provide immunoblot evidence for the purity of the stromal proteome isolated using our enhanced methods. In addition, we leverage our nanoliquid chromatography tandem mass spectrometry (nanoLC–MS/MS) data to assess the quality of our stromal proteome. Using strict criteria, proteins detected by 1 peptide spectral match, by one peptide, or were sporadically detected were designated as low-level contaminating proteins. A set of 254 proteins that reproducibly co-isolated with the tomato chloroplast stroma were identified. The subcellular localization, frequency of detection, normalized spectral abundance, and functions of the co-isolating proteins are discussed. Our optimized method for chloroplast isolation increased the yields of tomato chloroplasts eightfold enabling the proteomics analysis of the chloroplast stromal proteome. The set of 254 proteins that co-isolate with the chloroplast stroma provides opportunities for developing a better understanding of the extensive and dynamic interactions of chloroplasts with other organelles. These co-isolating proteins also have the potential for expanding our knowledge of proteins that are co-localized in multiple subcellular organelles.

中文翻译：

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方法：优化、高产的番茄叶叶绿体分离和基质提取方案，用于蛋白质组学分析和叶绿体共定位蛋白的鉴定

叶绿体是感知代谢和应激信号并将其传递给不同细胞成分的关键细胞器，同时仍然是光合作用和代谢工厂的所在地。完整叶子、叶绿体和质体亚细胞器部分的蛋白质组已在模型植物拟南芥中进行了评估，但很少有研究对作物中质体的蛋白质组进行表征。番茄（Solanum lycopersicum）是一种重要的世界作物，也是研究伤害、食草和果实成熟的模型系统。虽然在理解果实成熟过程中蛋白质组和代谢组变化方面已经取得了重大进展，但对番茄叶绿体蛋白质组或其子区室的了解却少之又少。为了了解叶绿体蛋白质组动力学响应应激的长期目标，我们描述了一种高产方法来分离完整的番茄叶绿体和基质蛋白以进行蛋白质组学研究。确定并修改了限制番茄叶绿体产量的参数以提高产量。与已发表的数据相比，我们的优化方法使叶绿体产量分别比已发表的菠菜和拟南芥叶方案提高了 6.7 倍和 4.3 倍；此外，番茄基质蛋白产量比拟南芥基质蛋白产量高出 79 倍。我们提供免疫印迹证据，证明使用我们的增强方法分离的基质蛋白质组的纯度。此外，我们利用纳米液体色谱串联质谱 (nanoLC-MS/MS) 数据来评估基质蛋白质组的质量。使用严格的标准，通过一种肽光谱匹配、一种肽检测到的蛋白质或偶尔检测到的蛋白质被指定为低水平污染蛋白质。鉴定出一组 254 种蛋白质，它们与番茄叶绿体基质可重复地共分离。讨论了共分离蛋白质的亚细胞定位、检测频率、归一化光谱丰度和功能。我们优化的叶绿体分离方法将番茄叶绿体的产量提高了八倍，从而能够对叶绿体基质蛋白质组进行蛋白质组学分析。与叶绿体基质共分离的 254 种蛋白质为更好地了解叶绿体与其他细胞器的广泛和动态相互作用提供了机会。这些共分离蛋白质还有可能扩展我们对共定位于多个亚细胞细胞器中的蛋白质的了解。